Diagnosis of neurodegenerative diseases

ABSTRACT

The invention relates to a method of diagnosis of vCJD in a diagnostic sample of a valid body tissue taken from a human subject, which comprises detecting an increased concentration of a protein in the diagnostic sample, compared with a sample of a control human subject, the protein being: beta-actin (SwissProt Acc. No. P60709), apolipoprotein A-IV precursor (SwissProt Acc. No. P06727); haptoglobin beta-chain consisting of residues 162-406 (SwissProt Acc. No. P00738); haemoglobin beta chain (SwissProt Acc. No. P02023); or alpha-1-antitrypsin (SwissProt Acc. No. P01009); or a decreased concentration of a protein in the diagnostic sample, compared with a sample of a control, normal human subject, the protein being plasma protease (C1) inhibitor precursor (SwissProt Acc. No. P05155); complement component 1, s sub-component (SwissProt Acc. No. P09871); butyrylcholinesterase precursor (SwissProt Acc. No. P06276); complement component C4B (SwissProt Acc. No. P01028); lumican (SwissProt Acc. No. P51884); alpha-fibrinogen precursor (SwissProt Acc. No. P02671); IGHG4 protein (Swiss Prot Acc. No. Q8TC63) or immunoglobulin lambda heavy chain. Other marker proteins are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the diagnosis of neurodegenerative diseases,namely variant Creutzfeld-Jakob Disease (vCJD).

2. Description of the Related Art

The neuropathology of Creutzfeld-Jakob disease, as for other priondiseases, manifests itself as a characteristic spongiform appearance ofthe brain tissue, neuronal cell death in the central nervous system,accompanied by astrocyte proliferation and in some cases the depositionof amyloid plaques. Characteristic to all prion diseases is theaccumulation in the brain of an altered, disease-associated form of thenormal prion protein (PrP^(C)) represented as PrP^(Sc). Four types ofPrP^(Sc) are associated with human prion disease. Of these, type 4 isassociated only with variant CJD (vCJD) which came to light in the UK in1996. It is believed to have arisen from the consumption by humans ofBSE-infected beef. No samples from other prion diseases have shown atype 4 profile.

The difficulties of diagnosis of vCJD, have led to the need for furthermethods to be developed.

SUMMARY OF THE INVENTION

The invention provides the use of specified marker proteins and theirpartners in or for the diagnosis of vCJD. These marker proteins havebeen found to be differentially expressed in two dimensionalelectrophoresis and/or Surface Enhanced Laser Desorption Ionisation(SELDI) time of flight mass spectrometry profiling of plasma.

The marker proteins and their differential expression characteristicsare as follows:

-   1A. Proteins present in an increased concentration in a vCJD sample    compared with neurological and/or non-diseased controls: haptoglobin    beta chain consisting of residues 162-406 (SwissProt Acc. No.    P00738); haemoglobin beta chain (SwissProt No. P02023),    alpha-1-antitrypsin (SwissProt Acc. No. P01009), beta-actin    (SwissProt Acc. No. P60709), haemoglobin beta chain (SwissProt Acc.    No. P02023) and apolipoprotein A-IV precursor (SwissProt Acc. No.    P06727);-   1B. Protein present in an decreased concentration in a vCJD sample,    compared with a control: alpha-fibrinogen precursor (SwissProt Acc.    No. P02671); IGHG4 protein (SwissProt Acc. No. Q8TC63);    immunoglobulin lambda heavy chain; plasma protease (C1) inhibitor    precursor (SwissProt Acc. No. P05155); complement component 1, s    sub-component (SwissProt Acc. No. P09871), butyrylcholinesterase    precursor (SwissProt Acc. No. P06276), complement component C4B    (SwissProt Acc. No. P01028), and lumican (SwissProt Acc. No. P51884)-   2. Proteins present in an increased or decreased concentration in a    vCJD sample compared with a control:

Protein ID Swiss Prot Accession # Transthyretin P02766 HaptoglobinP00738 Apolipoprotein C-III P02656 Vitronectin precursor P04004Hemoglobin beta chain P02025 IgG lambda chain C P01842 IgG kappa chain CP01834 Serum Albumin P02768 Apolipoprotein A-1 P02647 Actin P62736alpha-Fetuin P02765

Thus, the invention includes specifically:

-   1. A method of diagnosis of vCJD in a diagnostic sample of a valid    body tissue, especially a body fluid, taken from a human subject,    which comprises detecting an increased concentration of a protein in    the diagnostic sample, compared with a sample of a non    neurologically diseased control, normal human subject, the protein    being:-   haptoglobin beta-chain consisting of residues 162-406 (SwissProt    Acc. No. P00738);-   haemoglobin beta chain (SwissProt Acc. No. P02023);-   alpha-1-antitrypsin (SwissProt Acc. No. P01009);-   beta actin (SwissProt Acc. No. P60709) or-   apolipoprotein A-IV precursor (SwissProt Acc. No. P06727)-   or a decreased concentration of a protein in the diagnostic sample,    compared with a sample of a non-neurologically diseased control,    normal human subject, the protein being alpha-fibrinogen precursor    (SwissProt Acc. No. P02671);-   IGHG4 protein (Swiss Prot Acc. No. Q8TC63) or-   immunoglobulin lambda heavy chain.-   2. A method of diagnosis of vCJD in a diagnostic sample of a valid    body tissue, especially a body fluid, taken from a human subject,    which comprises detecting an increased concentration of a protein in    the diagnostic sample, compared with a sample of a control, normal    human subject, the protein being selected from:

Protein ID Swiss Prot Accession # Transthyretin P02766 HaptoglobinP00738 Apolipoprotein C-III P02656 Vitronectin precursor P04004Hemoglobin beta chain P02025 IgG lambda chain C P01842 IgG kappa chain CP01834 Serum Albumin P02768 Apolipoprotein A-1 P02647 Actin P62736alpha-Fetuin P02765

-   3. A method of diagnosis which distinguishes vCJD from other    neurological disease in a diagnostic sample of a valid body tissue,    especially a body fluid, taken from a human subject, which comprises    detecting an increased or decreased concentration of a protein in    the diagnostic sample, compared with a reference sample, the protein    being haemoglobin beta chain (SwissProt Acc. No. P02023), which is    increased in a vCJD sample compared with other neurological disease;    or a protein selected from: plasma protease (C1) inhibitor precursor    (SwissProt Acc. No. P05155); complement component 1, s sub-component    (SwissProt Acc. No. P09871), butyrylcholinesterase precursor    (SwissProt Acc. No. P06276), complement component C4B (SwissProt    Acc. No. P01028), and lumican (SwissProt Acc. No. P51884), which is    decreased in a vCJD sample compared with other neurological disease.

The marker protein can be present in the body tissue in any biologicallyrelevant form, e.g. in a glycosylated, phosphorylated, multimeric orprecursor form.

Although there is a high degree of confidence in the identification ofthe marker proteins specified above, the invention can be definedalternatively in terms of the proteins within the differentiallyexpressed spots on a two dimensional electrophoretic gel, namely thoseidentified in FIGS. 2 to 5 herein, without regard to the names anddatabase identifications given above.

DEFINITIONS

The term “differentially expressed” in the context of 2 dimensional gelelectrophoresis means that the stained protein-bearing spots are presentat a higher or lower optical density in the gel from the sample takenfor diagnosis (the “diagnostic sample”) than the gel from a control orother comparative sample, and in the context of SELDI-TOF means that theprotein peak is at a higher or lower intensity in the mass spectrogramfrom the sample taken for diagnosis (the “diagnostic sample”) than themass spectrogram from a control or other comparative sample. It followsthat the proteins are present in the plasma of the diagnostic sample ata higher or lower concentration than in the control or other comparativesample in the same direction of differential expression seen in Sdimensional gel electrophoresis and SELDI-TOF.

The term “control” refers to a human subject not suffering from vCJD.

The terminology “increased/decreased concentration . . . compared with asample of a control” does not imply that a step of comparing is actuallyundertaken, since in many cases it will be obvious to the skilledpractitioner that the concentration is abnormally high. Further, whenthe stage of vCJD progression is being monitored progressively, thecomparison made can be with the concentration previously seen in thesame subject earlier in the progression of the disease.

The term “binding partner” includes a substance that recognises or hasaffinity for the marker protein. It may or may not itself be labelled.

The term “marker protein” includes all biologically relevant forms ofthe protein identified.

The term “diagnosis”, as used herein, includes determining whether vCJDis present or absent and also includes determining the stage to which ithas progressed. The diagnosis can serve as the basis of a prognosis asto the future outcome for the patient.

The term “valid body tissue” means any tissue in which it may reasonablybe expected that a marker protein would accumulate in relation to vCJD.While it will principally be a body fluid, it also includes brain ornerve tissue, tonsil, spleen and other lymphoreticular tissue, it beingunderstood that the diagnosis can be made either pre-mortem or postmortem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph of a typical two dimensional gel performed foranalytical purposes, by the method described in Example 1 below, on asample derived from a vCJD patient. The molecular weight (relativemolecular mass) is shown on the ordinate in kiloDaltons. Molecularweight markers are shown at the left-hand side. The isoelectric point(pI) is shown on the ordinate, increasing from left to right.

FIG. 2 is a photograph of a similar gel, but marked with spots 1713,1893, 1960, 2730 and 2732, explained in detail in Example 1. Spot 1960,although a marker protein for HD (not vCJD) is shown here forconvenience, since it does appear in vCJD patients, at about the samelevel as in a control.

FIG. 3 is a photograph enlarged to show a portion of the gel of FIG. 1and the spots 846 and 1526.

FIG. 4 is a photograph enlarged to show another portion of the gel ofFIG. 1 and the spot 1488. The spots 1293 and 2885 are also shown, butthey were not further pursued, as explained in Example 1.

FIG. 5 is similar to FIG. 2, but showing spots 1713 and 1960 in a samplederived from an HD patient.

FIGS. 6 to 13 are SELDI traces, as described more fully in Example 2.

FIG. 14 is an image of a silver stained gel of the material extractedfrom depleted plasma Q10 chips, as described in Example 2.

FIG. 15 is an image of a silver stained gel of the material extractedfrom depleted plasma WCX CM10 chips, as described in Example 2.

DESCRIPTION OF PREFERRED EMBODIMENTS

A preferred method of diagnosis comprises performing a binding assay forthe marker protein. Any reasonably specific binding partner can be used.Preferably the binding partner is labelled. Preferably the assay is animmunoassay, especially between the marker and an antibody thatrecognises the protein, especially a labelled antibody. It can be anantibody raised against part or all of it, most preferably a monoclonalantibody or a polyclonal anti-human antiserum of high specificity forthe marker protein.

Thus, the marker proteins described above are useful for the purpose ofraising antibodies thereto which can be used to detect the increased ordecreased concentration of the marker proteins present in a diagnosticsample. Such antibodies can be raised by any of the methods well knownin the immunodiagnostics field.

The antibodies may be anti- to any biologically relevant state of theprotein. Thus, for example, they could be raised against theunglycosylated form of a protein which exists in the body in aglycosylated form, against a more mature form of a precursor protein,e.g. minus its signal sequence, or against a peptide carrying a relevantepitope of the marker protein.

The sample can be taken from any valid body tissue, especially bodyfluid, of a (human) subject, but preferably blood, plasma or serum.Other usable body fluids include cerebrospinal fluid (CSF), urine andtears.

According to another embodiment of the invention, the diagnosis iscarried out pre- or post mortem on a body tissue of neurological originrelevant to vCJD such as from the brain or nerves, tonsil, spleen orother lymphoreticular tissue. The tissue is pre-treated to extractproteins therefrom, including those that would be present in the bloodof the deceased, so as to ensure that the relevant marker proteinsspecified above will be present in a positive sample. For the purposesof this patent specification, such an extract is equivalent to a bodyfluid.

By way of example, brain tissue is dissected and sub-sectionssolubilised by methods well established in the art such as mechanicaldisruption in a phosphate buffered saline, in a ratio of about 100 mgtissue to 1 ml buffer. Where desirable chaotropic salts such asguanidinium hydrochloride or sodium dodecylsulphate may be included toinactivate the infectious prion agent so long as this does not interferewith subsequent detection of the vCJD biomarkers.

The preferred immunoassay is carried out by measuring the extent of theprotein/antibody interaction. Any known method of immunoassay may beused. A sandwich assay is preferred. In this method, a first antibody tothe marker protein is bound to the solid phase such as a well of aplastics microtitre plate, and incubated with the sample and with alabelled second antibody specific to the protein to be assayed.Alternatively, an antibody capture assay could be used. Here, the testsample is allowed to bind to a solid phase, and the anti-marker proteinantibody is then added and allowed to bind. After washing away unboundmaterial, the amount of antibody bound to the solid phase is determinedusing a labelled second antibody, anti- to the first.

In another embodiment, a competition assay is performed between thesample and a labelled marker protein or a peptide derived therefrom,these two antigens being in competition for a limited amount ofanti-marker protein antibody bound to a solid support. The labelledmarker protein or peptide thereof could be pre-incubated with theantibody on the solid phase, whereby the marker protein in the sampledisplaces part of the marker protein or peptide thereof bound to theantibody.

In yet another embodiment, the two antigens are allowed to compete in asingle co-incubation with the antibody. After removal of unbound antigenfrom the support by washing, the amount of label attached to the supportis determined and the amount of protein in the sample is measured byreference to standard titration curves established previously.

The label is preferably an enzyme. The substrate for the enzyme may be,for example, colour-forming, fluorescent or chemiluminescent.

The binding partner in the binding assay is preferably a labelledspecific binding partner, but not necessarily an antibody. For example,when the marker protein is alpha-1-antitrypsin, the specific bindingpartner can be trypsin. The binding partner will usually be labelleditself, but alternatively it may be detected by a secondary reaction inwhich a signal is generated, e.g. from another labelled substance.

It is highly preferable to use an amplified form of assay, whereby anenhanced “signal” is produced from a relatively low level of protein tobe detected. One particular form of amplified immunoassay is enhancedchemiluminescent assay. Conveniently, the antibody is labelled withhorseradish peroxidase, which participates in a chemiluminescentreaction with luminol, a peroxide substrate and a compound whichenhances the intensity and duration of the emitted light, typically4-iodophenol or 4-hydroxycinnamic acid.

Another preferred form of amplified immunoassay is immuno-PCR. In thistechnique, the antibody is covalently linked to a molecule of arbitraryDNA comprising PCR primers, whereby the DNA with the antibody attachedto it is amplified by the polymerase chain reaction. See E. R.Hendrickson et al., Nucleic Acids Research 23: 522-529 (1995). Thesignal is read out as before.

Alternatively, the diagnostic sample can be subjected to two dimensionalgel electrophoresis to yield a stained gel and the increased ordecreased concentration of the protein detected by an increased anincreased or decreased intensity of a protein-containing spot on thestained gel, compared with a corresponding control or comparative gel.The relevant spots, diseases identified and differential expression arethose listed in Table 1 below. The invention includes such a method,independently of the marker protein identification given above and inTable 2.

The diagnosis does not necessarily require a step of comparison of theconcentration of the protein with a control, but it can be carried outwith reference either to a control or a comparative sample. Thus theinvention can be used to determine the stage of progression of vCJD ifdesired by comparison of protein levels with results obtained earlierfrom the same patient or by reference to standard values that areconsidered typical of the stage of the disease. In this way, theinvention can be used to determine whether, for example after treatmentof the patient with a drug or candidate drug, the disease has progressedor not. The result can lead to a prognosis of the outcome of thedisease.

The invention further includes the use for a diagnostic (and thuspossibly prognostic) or therapeutic purpose of a partner material whichrecognises, binds to or has affinity for a marker protein specifiedabove and/or represented by a differentially expressed two dimensionalgel electrophoretic spot shown in any of FIGS. 2 to 5 herein, or thedifferentially expressed SELDI peaks at MW 3223 Da, MW4132 Da, MW4340Da, MW4490 Da, MW6243 Da, MW 7533 Da, MW 8644 Da, MW 8856 Da, MW 8868Da, MW 14257 Da, MW 27202 Da. Thus, for example, antibodies to themarker proteins, appropriately humanised where necessary, may be used totreat vCJD and HD. The partner material will usually be an antibody andused in any assay-compatible format, conveniently an immobilised format,e.g. micro- or nano-particle beads or a glass, silicone ornitrocellulose chip. Either the partner material will be labelled or itwill be capable of interacting with a label.

The invention further includes a kit for use in a method of diagnosis,which comprises a partner material, as described above, in anassay-compatible format, as described above, for interaction with aprotein present in the diagnostic sample.

The diagnosis can be based on the differential expression of one, two,three or more of the marker proteins. Further, it can be part of a widerdiagnosis in which one or more additional diseases are diagnosed inaddition to vCJD. Accordingly vCJD can be diagnosed along with at leastone other disease, which may or may not be neurological, in the samesample of body fluid, by a method which includes detecting an increasedconcentration of another protein in the diagnostic sample, compared witha sample of a control, normal human subject. These other disease(s) canbe any which are diagnosable in a body fluid. They may be neurological,e.g. another transmissible spongiform encephalopathy, Alzheimer'sdisease, Huntington's disease, Parkinson's Disease, meningitis, but arenot necessarily neurological, for example toxic shock syndrome, MRSA orCeliac disease.

Thus, in particular, it is contemplated within the invention to use anantibody chip or array of chips, capable of diagnosing one or moreproteins that interact with that antibody.

The following Examples illustrate the invention.

Example 1

Ten plasma samples were taken from patients (4 female, 6 male) who werediagnosed with variant CJD (vCJD), ten from patients (7 female, 3 male)diagnosed by genetic testing as having Huntington's Disease (HD) servingas a neurological disease control and ten from non-diseased controls,i.e. normal patients (8 female, 2 male) not having any neuropathologicalsymptoms.

Albumin and IgG were removed from the samples using a kit supplied byAmersham Biosciences UK Ltd. This kit contains an affinity resincontaining antibody that specifically removes albumin and IgG directlyfrom whole human serum and plasma samples. It is claimed that more than95% albumin and more than 90% IgG removal from 15 μl human serum/plasmacan be achieved, thereby increasing the resolution of lower abundanceproteins in subsequent electrophoresis. A microspin column is used,through which the unbound protein is eluted.

Depletion was carried out according to the manufacturer's instructionsusing a starting volume of 15 μl of crude plasma sample. The resin wasadded to the plasma, the mixture incubated with shaking, transferred toa microspin column, centrifuged and the filtrate collected. Theresulting depleted sample was concentrated and de-salted by acetoneprecipitation (as recommended in the instructions of the kit). Theacetone was decanted and the pellets were re-suspended in standard 2-Dgel lysis buffer (9.5 M urea, 2% CHAPS, 1% DTT, 0.8% Pharmalyte, pH3-10, protease inhibitors (1 tablet/10 ml lysis buffer) (Roche). Thissuspension was used for the two dimensional gel electrophoresis.

Two dimensional gel electrophoresis was performed according to J. Weekeset al., Electrophoresis 20: 898-906 (1999) and M. Y. Heinke et al.,Electrophoresis 20: 2086-2093 (1999), using 18 cm immobilised pH 3-10non-linear gradient strips (IPGs). The second dimension was performedusing 12% T SDS polyacrylamide gel electrophoresis. For the initialanalysis, the gels were loaded with 75 micrograms of protein. The gelswere silver-stained with the analytical OWL silver stain (InsightBiotechnologies, UK).

Quantitative and qualitative image analysis was performed using thesoftware Progenesis™ Workstation, version 2003.02 (Nonlinear DynamicsLtd.). The images were processed through the automatic wizard for spotdetection, warping and matching. Thereafter, all images underwentextensive manual editing and optimal matching to the reference gel (>80%per gel). Following background subtraction and normalisation to totalspot volume, protein spot data was exported to Excel for quantitativestatistical analysis and comparisons of qualitative changes.

The student t-test, at the 95% confidence interval, was performed forevery protein spot that could be compared between the samples from thediseased patients and the controls and which was present in at least 60%of the gels of each group, i.e. at least 6. A log transformation wasperformed, since this gave a more normal distribution, thus bettermeeting the assumptions of this test as applied to independent samples.

The spots for which a significant increase or decrease was observed incomparisons between the three groups are shown in FIGS. 2 to 5 andlisted in Table 1.

TABLE 1 Quantitative change No. samples (Increase/Decrease in intensityof in which spot Spot spot in comparisons between p value seen vCJD/ No.FIG. vCJD, HD and control samples). (t-test) HD/Control 1893 2 Inc. vCJDvs. Control 0.001   10/10/10 2732 2 Inc. vCJD vs. Control 0.001  10/10/10 2732 2 Inc. vCJD vs. Neurological Control 0.002   10/10/10 17132 Inc. vCJD vs. Control 0.003   8/10/6 1713 5 Inc. HD vs. Control0.000065 8/10/6 1526 3 Inc. vCJD vs. Control 0.003   10/10/8 1488 4 Dec.vCJD vs. Control 0.003   7/10/10 2730 2 Inc. vCJD vs. Control 0.003  10/9/10 846 3 Dec. vCJD vs. Neurological 0.006   9/8/6 Control 1960 5Inc. HD vs. Control 0.004   10/10/10

Quantitative changes were seen in two other spots (1293 and 2885) on theanalytical gels, but not on the preparative gels (see below). These wereboth in the vCJD vs. neurological control (HD) comparison. Spot 1293 wasdecreased and 2885 increased in vCJD versus neurological control (HD).

It will be seen that spot 1713 is one to which particularly highconfidence in the results can be attached in relation to the increase inits intensity in the neurological control (HD) samples versus controls.This spot also showed an increase in the vCJD vs. control comparison.

Spots 1893, 2732, 1526 and 2730 showed increases in the vCJD versuscontrols comparison. Spot 2732 also showed an increase in the vCJDsamples compared with neurological control (HD).

Spot 846 was decreased in the vCJD samples compared with neurologicalcontrol (HD).

For preparative purposes, further two dimensional gels were then made bythe same method, by pooling all samples within each experimental groupand loading the gels with 400 micrograms of protein. There were thusthree gels prepared, one for each group, which were silver stained,using PlusOne silver stain (Amersham Pharmacia Biosciences UK Ltd.).

Normally, the spots were excised from the preparative gels in which theywere elevated in intensity, but where this was not possible i.e. wherespots were decreased in intensity in vCJD, they were excised fromanother gel. After in-gel reduction, alkylation and digestion of theexcised material with trypsin, the peptides produced were extracted andsubsequently analysed by LC/MS/MS. This procedure involves separation ofthe peptides by reversed phase HPLC, followed by electrospraying toionise the sample, as it enters a tandem mass spectrometer. The massspectrometer records the mass to charge ratio of the peptide precursorions, which are then individually selected for fragmentation viacollisionally induced dissociation (CID). This so-called MS/MS scanallows for the sequence of the peptide to be determined. For eachsample, therefore, the data set includes accurately determined molecularweights for multiple peptides present, accompanied by correspondingsequence information. This is then used to identify the protein bysearching databases. In the present case, the Mascot search algorithmwas used against the National Center for Biotechnology Information(NCBI) non-redundant protein (nr) and SWISS-PROT databases.

The results of the identification are shown in Table 2. All the spots ofTable 1 that were differentially expressed on the gel were identified asknown proteins.

The Table shows the geninfo (gi) numbers of the NCBI database andSwissProt Accession numbers.

In some instances more than one protein was identified, which signifiesthat the spot excised contained a mixture of proteins, at least one ofwhich was differentially expressed on the gel. The proteins identifiedin the database had different molecular weights and isoelectric points,lower or higher, from those evident on the gel. This is entirely usualand can be accounted for by the protein within the gel spot havingundergone enzymatic or chemical cleavage or by having beenpost-translationally modified such as by glycosylation, phosphorylationor the addition of lipids.

As between spots 2730 and 2732, which relate to forms of the sameprotein, 2732 is of slightly higher pI and reference thereto should beunderstood accordingly.

TABLE 2 MW (DA) pI NCBI nr and No. peptides Spot from from HumanSwissProt Acc. matched (% No. gel gel protein identified No. coverage)1893 35473 5.30 Haptoglobin beta chain* gi/67586 15 (47%) P00738 273213387 7.07 Haemoglobin beta chain gi/4504349 13 (84%) P02023 1713 431085.19 Beta actin gi/4501885 14 (47%) P60709 Apolipoprotein A-IVgi/4502151  7 (26%) precursor P06727 1526 53638 4.74 Alpha-1-antitrypsingi/177827 3 (9%) P01009 1488 56468 7.32 Alpha-fibrinogen gi/182424 4(7%) precursor P02671 IGHG4 protein gi/19684073  4 (11%) Q8TC63Immunoglobulin lambda gi/2765425   5 (14%)** heavy chain 2730 13470 6.88Haemoglobin beta chain gi/4504349 10 (84%) P02023 846 93995 4.76 Plasmaprotease (C1) gi/179619  5 (11%) inhibitor precursor P05155 Complementcomponent 1, gi/34785163PP09871 2 (5%) s sub-componentButyrylcholinester-ase gi/4557351 2 (5%) precursor P06276 Complementcomponent gi/187771 1 (6%) C4B P01028 Lumican gi/642534 1 (4%) P51884Footnotes to Table 2 *the haptoglobin beta-chain consisting of residues162-406 of the database sequence, which is formed by enzymatic cleavageof the precursor; this protein is glycosylated in plasma **including 2unique peptides

Example 2 Discovery of vCJD Biomarkers by SELDI-TOF Mass Spectrometry

A second set of vCJD biomarkers were revealed using Surface EnhancedLaser Desorption Ionisation (SELDI) time of flight mass spectrometry.Experiments to establish the identity of these new candidates are alsodescribed.

1.1 Sample Preparation for SELDI Discovery

The plasma samples used in Example 1 from clinically confirmed cases ofvCJD (n=10), neurological controls (HD) (n=10) and non-diseased control(n=10) patients were collected from the MRC Prion unit. Two microlitersof each of the depleted samples were diluted in 3 ul of lysis buffercontaining 9.5 M urea, 2% CHAPS, 0.8% pharmalyte pH 3-10, 1% DTT andprotease inhibitor and undepleted samples were diluted in the same ratiousing the above lysis buffer without pharmalyte.

1.2 Plasma Depletion

Consistent with the previous 2DE study, Albumin and IgG were removedfrom the plasma using a commercially available resin (GE Healthcare).This kit is antibody based and contains a resin that specificallyremoves albumin and IgG directly from whole human serum and plasmasamples. It is claimed that >95% albumin and >90% IgG from 15 μl humanserum/plasma can be achieved, thereby increasing the resolution of lowerabundance proteins. A microspin column is used through which the unboundprotein is eluted.

Depletion was carried out according to the manufacturer's instructionsusing a starting volume of 15 μl of crude plasma sample. The resultingdepleted sample was acetone precipitated (as recommended in theinstructions of the kit) and re-suspended in standard 2DE lysis buffer(as indicated in section 2.2 above)

1.3 Surface Enhanced Laser Desorption Ionisation (SELDI) MassSpectrometry

Profiling of depleted plasma samples were performed using an eight spotstrong anion exchange (Q10) protein chip array and profiling ofundepleted plasma were performed using both the eight spot Q10 and weakcation exchange (CM10) protein chip arrays. All samples were run induplicate and in a randomised manner. Essentially, all the Q10 and CM10chips were equilibrated four times in the appropriate wash buffer. ForQ10 chips, 100 mM Tris HCl pH 9.0 was used as the wash buffer and forCM10 the wash buffer was 50 mM sodium acetate pH 7.5. 5 μl of thediluted samples were applied to each spot and this was then incubated ina humidity chamber for 45 minutes. Samples were carefully removed andthe chips were washed four times in the appropriate wash buffer and onewash with 18.2 MΩ water. 0.6 μl matrix solution containing 20 mg/mlsinnapinic acid (Ciphergen) in 50% acetonitrile (Fisher Scientific) and0.5% trifluroacetic acid was applied twice to each spots. Dataacquisition was performed using a PBS-II reader (Ciphergen Biosystems).Spectra were acquired using a summation of 155 shots with a laserintensity of 200, detector sensitivity of 8 and a focus mass m/z 25000.Baseline subtraction and normalisation on total ion count were performedon all the spectra. Internal calibration of each spectra was undertakenusing a minimum of 2 peaks in each spectrum.

SELDI traces for the depleted plasma Q10 SAX2 dataset are shown in FIGS.6 and 7. FIG. 6 shows SELDI spectra showing peaks in the region of m/z4100-4500. FIG. 7 shows SELDI spectra showing peaks in the region of m/z8600-9400. The upper and lower panels show overlayed spectra belongingto the control (CTRL) and vCJD groups, respectively. Asterisks (*) markthe peaks of interest.

SELDI traces for the undepleted plasma CM10 WCX dataset are shown inFIGS. 8 to 13.

1.4 SELDI Data Analysis

The data analysis approach adopted for this study comprises severalmodules as described below. To be considered as a candidate of interest,each biomarker must satisfy the following three criteria, the values ofwhich are derived either from the multivariate modelling process orunivariate tests.

-   The position of the peak of interest within the loadings plot    indicates an obvious contribution to the separation of the groups in    the data modelling process and this also survives a cross validation    exercise.-   A p value of <0.05 is achieved using a Mann-Whitney univariate test.-   The magnitude of change in abundance of the marker between two    groups is >1.5 fold either up or down regulated.    Pre-Processing:

All data were imported to the SIMCA-P software package (Umetrics).Variables corresponding to masses below m/z 2,500 were excluded due tothe considerable chemical noise in this region. The remaining variablescorresponding to masses between m/z 2,500 and m/z 100,000 were centeredto the mean value and Pareto scaled.

Principal Component Analysis (PCA):

PCA models were fitted to the data sets with as many components (A) aswould fit following the internal rules SIMCA-P uses to determine thesignificance of the components (Eriksson et al. 2001). The goodness offit (R²) and goodness of prediction (Q²) parameters were used to assessthe usefulness of each of the subsequent components fitted in the model.The automatically fitted components were inspected and kept as long asthe Q² parameter was increasing. The cumulative R² parameter for thefinal accepted component gave the total proportion of variance in thedata explained by the model. Plots were produced displaying theobservation scores (t) and variable loadings (p) for pairs of principalcomponents (a). The scores plots were inspected to look for patterns ofsystematic variation and outlying observations that could hamper laterclassification efforts. In particular, the positions of observationsanalysed on each chip were scrutinised to check for unusual chips. Thereproducibility of duplicated sample analyses were also checked usingthe scores plots. The Ellipse shown on the scores plots corresponds toHotelling's T² at 95%, a multivariate adaptation of a confidence region.For a data set with a multivariate normal distribution, 95% of theobservations would be expected to lie within the region encompassed bythe Ellipse, thus observations that are a long way outside the ellipsemay represent problems to be investigated and addressed.

Trends found through inspection of the scores plots were interpretedthrough inspection of the variables found on the corresponding loadingsplots. Individual m/z values plotted at the extremes of the plot wereconsidered to be most influential on the separation of the groups.Interestingly, such plots tend to show several consecutive m/zdatapoints, which effectively describe the original peak observed in theSELDI profiles themselves.

Partial Least Squares Data Analysis (PLS-DA) and Modelling:

Components (A) of PLS-DA models were fitted to the data sets as long asthey met the criteria used by SIMCA-P to determine the significance ofcomponents (Eriksson et al. 2001). As for the PCA modelling, the R² andQ² parameters were inspected to determine which components should beincluded in the model. Unlike the PCA modeling, PLS-DA models posses R²values describing the fit of the model to both the X (measurement)variables and the Y (class) variables. Plots were produced displayingthe observation scores (t) and the variable weights (w*c) for pairs ofPLS components (a). Because each PLS component is fitted so as to bothapproximate the X and Y data well and maximize the correlation betweenthe X and Y data, in practice the first one or two components usuallyseparate the observations well when there are few groups present in thedata set. The interpretation of the PLS scores and weights plots issimilar to that used to interpret a PCA model, with the PLS weightsbeing analogous to the PCA loadings. Hotelling's T² was computed anddisplayed on all PLS scores plots to help identify deviatingobservations.

The two parameters referred to as variable influence on projection (VIP)and PLS coefficients (COEFF) were used to determine which of all themasses measured in the SELDI spectral data were most important indefining the model parameters and explaining the groups. Specificthresholds were determined empirically and used to exclude thosevariables with VIP and COEFF values lower than the threshold. Theability of the PLS-DA models generated to correctly predict the class of(new) samples was determined by 2-fold cross-validation.Cross-validation was performed by dividing the data set into a trainingand a test set. A PLS-DA model was fitted to the training portion of thedata set and subsequently used to predict the classes of the testportion of the data set. The training and test data sets were thenswapped and the process repeated. The number of correct and incorrectclassifications from both rounds of testing were recorded and used tocalculate sensitivities and specificities of the predictions. Thiscross-validation method was used to test both the models built using thedata set containing all variables and those built following variableselection (as described above).

Univariate Methods:

Statistical significance testing was performed using the Protein Chipsoftware (Ciphergen Biosystems). Mann-Whitney (Wilcoxon) tests for twoindependent samples were used. Peak detection and matching wereperformed using the Protein Chip software and this data was thensubmitted to the Biomarker Wizard module for analysis. The p-value wastaken as the result of the test. The data for each of the marked peakswas also exported to Excel (Microsoft) as peak intensities to calculatethe fold change criteria for each peak. Because of the skeweddistributions observed for the areas or intensities of each set ofmatched peaks, the data were log₁₀ transformed prior to calculation ofthe mean and median values of the distributions as well as the standarddeviations. The parameters of the distributions were then transformedback onto the original scales in order to calculate fold-changes andeffect sizes. Fold-changes were calculated by dividing the larger of themean (or median) values by the smaller value of two groups, yielding avalue greater than or equal to one. Effect size (Cohen's D) wascalculated as the difference between the mean values of two groupsdivided by the pooled standard deviation.

1.5 Candidate Identification

Having produced/created a list of candidate peaks of interestcorresponding to each chip surface, the identity of the proteinsresponsible for each discriminating peak was determined.

Material was extracted directly from the chip surface and followingelectrophoretic separation and enzymatic digestion proteins wereidentified by electrospray tandem mass spectrometry (LC/MS/MS).

There are several advantages inherent to this strategy:

-   Pooling of material from several target positions overcomes    challenges working with low level of protein and increased    sensitivity is achieved.-   SDS-PAGE provides an additional stage of visualisation of the sample    as well as serving as an important separation and concentration    step.-   LC/MS/MS of digested proteins is routine methodology in our    laboratory.

Bands of interest were excised from the silver stained gel and “in-gel”reduction, alkylation and digestion with trypsin were performed prior tosubsequent analysis by LC/MS/MS. Peptides were extracted from the gelpieces by a series of acetonitrile and ammonium bicarbonate washes. Theextract was pooled with the initial supernatant and lyophilised. Eachsample was then resuspended in 23 μl of 50 mM ammonium bicarbonate.Chromatographic separations were performed using an Ultimate LC system(Dionex, UK). Peptides were resolved by reversed phase chromatography ona 75 μm C18 PepMap column. A gradient of acetonitrile in 0.05% formicacid was delivered to elute the peptides at a flow rate of 200 nl/min.Peptides were ionised by electrospray ionisation using a Z-spray sourcefitted to a QTof-micro (Waters Corp.). The instrument was set to run inautomated switching mode, selecting precursor ions based on their m/zand intensity, for sequencing by collision-induced fragmentation.

The mass spectral data was processed into peak lists (containing theprecursor ion m/z and charge state and the m/z and intensity of thefragment ions. Database searching was undertaken to establish theidentity of the protein(s) present. This was performed using the Mascotsearch algorithm against the NCBI non-redundant (nr) and SWISS-PROTdatabases.

Once proteins were identified the expected molecular weight of themature proteins was extrapolated from the information contained withinthe database entry and correlated with the molecular weight determinedexperimentally in the original SELDI profiles. In this way it waspossible in most cases to assign related species to a single proteinsequence.

2.1 SELDI Data Analysis

Following extensive analysis using multivariate techniques andMann-Whitney tests, the depleted plasma study (Q10 SAX chip) revealedvariation in several peaks, which discriminate between vCJD and controlsamples (see Table 3 below). Similarly, for undepleted plasma the CM10WCX profiles reveal additional discriminatory peaks (see Table 4 below).

TABLE 3 SELDI peaks of interest discriminating between vCJD and controlsamples (depleted plasma study using Q10 SAX chip) Candidate Fold- Fold-Reference Peak of p- Change change Direction Cohen's number Interestvalue^(a) (mean)^(b) (median)^(c) of change D^(d) P1 8644 0.023 2.473.19 Decreased 1.130 P2 8856 0.045 1.75 1.95 In CJD 0.934 P3 4132 0.0013.87 5.16 Increased 1.743 P4 4340 0.011 1.85 1.93 in CJD 1.136 P5 44900.023 1.69 1.76 0.990

TABLE 4 SELDI peaks of interest discriminating between vCJD and controlsamples (undepleted plasma study using CM10 WCX chip) Candidate Fold-Fold- Reference Peak of p- Change change Direction Cohen's numberInterest value^(a) (mean)^(b) (median)^(c) of change D^(d) P6 3223 0.0091.50 1.50 Decreased −1.300 P7 8868 0.023 1.60 1.20 In CJD −2.100 P827202 0.037 1.50 1.50 −1.500 P9 6243 0.023 1.50 1.50 Increased 3.000 P107533 0.003 2.40 2.40 in CJD 1.900 P11 14257 0.028 1.80 1.70 0.900 Notes:^(a)p-values computed for a Mann-Whitney test (not corrected formultiple testing). ^(b)Mean and median peak intensity values for eachgroup were estimated after log₁₀ transformation of the data. Theestimates were transformed back to the original scale prior tocalculating fold-changes. ^(c)The effect size (Cohen's D) is computed asthe different between the means divided by the pooled standarddeviation.

2.2 Candidate Identification

The silver stained gel of the material extracted from the depletedplasma Q10 chips is shown in FIG. 14. Although a total of 36 sectionswere excised, we considered 9 sections to be important to the keyobjective of the identification of the proteins responsible for thepeaks indicated in Table 3. Hence, we gave priority to the LC/MS/MSanalysis of these particular bands namely bands 2, 3, 4, 5, 7 and 8 fromthe control lane and bands 9, 10 and 11 from the vCJD lane. A summary ofthe results is given in Table 5. Similarly, LC/MS/MS was also undertakenon material extracted from the CM10 WCX chip. The silver stained gel ofthe material extracted from the depleted plasma WCX CM10 chips is shownin FIG. 15 and the proteins identified in the analyses are shown inTable 6.

TABLE 5 Summary of LC/MS/MS results for Bands extracted from the Q10chips Mr Experi- Swiss Prot indicated in mental Accession database entryBand# Mr (kDa) Protein ID # (kDa)* 2 12 Transthyretin P02766 15.8 3 10Haptoglobin P00738 45.1 4 9 Apolipoprotein C-III P02656 10.8 5 8Apolipoprotein C-III P02656 10.8 7 10 Vitronectin precursor P04004 54.2Hemoglobin beta chain P02025 15.8 8 6 Apolipoprotein C-III P02656 10.8 9 vCJD 5 Not yet established — — 10 vCJD 4-5 Not yet established — — 11vCJD 3 Not yet established — — Note: *The molecular weights indicated inSwiss Prot generally refer to the precursor proteins rather than themature proteins, which exist after processing. Please also note thatfurther interpretation of the LC/MS/MS data for Bands 9, 10 and 11 mayreveal the presence of unexpected proteolytic fragments.

TABLE 6 Summary of LC/MS/MS results for Bands extracted from the CM10chips Mr Swiss Prot indicated in Experimental Accession database entryBand# Mr (kDa) Protein ID # (kDa)* 1 + 6 27  IgG lambda chain C P0184211 14-16 17-23 IgG kappa chain C P01834 11 Serum Albumin P02768 69 32-3417-23 IgG kappa chain C P01834 11 Apolipoprotein A-1 P02647 30 19-2112-14 Serum Albumin P02768 69 37-39 12-14 Serum Albumin P02768 69 22-24 9-12 Serum Albumin P02768 69 Apolipoprotein C-III P02656 10 40-42  9-12Actin P62736 41 3 + 8 9 Serum Albumin P02768 69 4 + 9 9 Serum AlbuminP02768 69  5 + 10 3 Serum Albumin P02768 69 alpha-Fetuin P02765 39

The results suggest that a collection of human albumin fragments existin the SELDI profiles and that these differ in abundance when vCJD casesare compared to controls. It is apparent that these relate to the Nterminal region of the protein in particular. We therefore claim thatsix candidate peaks are related to N-terminal fragments of Human albuminand the basis of this claim is illustrated in Table 7 below.

TABLE 7 List of Candidate biomarkers matched to fragments within theN-terminal region of Human Albumin Candidate [M + H]⁺ Expected Ref#observed m/z Average Mr Residues % Error P1 8644 8642 2-78  0.020 P28856 8857 2-80  0.010 P3 4132 4130 41-78  0.050 P4 4340 4344 41-80 0.090 P11 14257 14255 5-129 0.010 P8 27202 27206 6-242 0.015

The sequence of Human Albumin precursor was retrieved from the SwissProt database (P02768) and exported into the Biolynx software packagewithin MassLynx for examination. The Mature albumin sequence is createdby removing the first 18 amino acids as the signal peptide as well as afurther 5 amino acids which relate to a pro peptide sequence. Theresidue numbers indicated refer to the mature protein of 585 amino acidsin total. Each observed average Mr value is within 0.1% mass error ofthe predicted value.

For Hemoglobin beta chain (P02025) we can match a fragment extendingfrom residues 4-43 to the peak at m/z 4490 (P5) and this encompasses twopeptides observed in the LC/MS/MS data. These results are summarisedbelow. Therefore we claim that candidate reference number P5 is likelyto be residues 4-43 of Hemoglobin beta chain.

The potential processing of Hemoglobin beta chain is shown as follows:

The amino acid sequence of Hemoglobin beta chain is shown with thelocation of potential fragment, residues 4-43 (P5), indicated by thearrow. The box indicates the location of the peptides observed in theLC/MS/MS data.

Each of the above-cited publications and database references is hereinincorporated by reference to the extent to which it is relied on herein.

The invention claimed is:
 1. A method comprising: providing a blood, serum, or plasma sample from a human subject; subjecting the sample to two dimensional gel electrophoresis to yield a stained gel; and quantifying an increased or decreased concentration of each of a plurality of proteins in the sample, compared with a sample of a control, normal human subject, the plurality of proteins having an increased concentration being selected from the group consisting of: beta-actin (SwissProt Acc. No. P60709); apolipoprotein A-IV precursor (SwissProt Acc. No. P06727); haptoglobin beta-chain consisting of residues 162-406 (SwissProt Acc. No. P00738); hemoglobin beta chain (SwissProt Acc. No. P02023); and alpha-1-antitrypsin (SwissProt Acc. No. P01009); and the plurality proteins having a decreased concentration being selected from the group consisting of: plasma protease (C1) inhibitor precursor (SwissProt Acc. No. P05155); complement component 1, s sub-component (SwissProt Acc. No. P09871); butyrylcholinesterase precursor (SwissProt Acc. No. P06276); complement component C4B (SwissProt Acc. No. P01028); lumican (SwissProt Acc. No. P51884); alpha-fibrinogen precursor (SwissProt Acc. No. P02671); IGHG4 protein (Swiss Prot Acc. No. Q8TC63); and immunoglobulin lambda heavy chain, wherein the increased or decreased concentration of each of the plurality of proteins is detected by an increased or decreased intensity of a protein-containing spot on the stained gel, compared with a corresponding control gel.
 2. The method according to claim 1, wherein the detecting step further comprises performing on the sample a binding assay for the protein.
 3. The method according to claim 2, wherein the binding assay comprises causing the protein of the sample to interact with a specific binding partner and detecting the interaction.
 4. The method according to claim 3, wherein the labelled specific binding partner is a labelled antibody that recognizes the protein.
 5. The method according to claim 1, wherein at least one of the plurality of proteins detected is an increased intensity of haptoglobin beta chain, Hemoglobin beta chain, or alpha-1-antitrypsin; or a decreased intensity of alpha-fibrinogen precursor, IGHG4 protein, or Immunoglobulin lambda heavy chain.
 6. The method of claim 1, wherein samples taken at intervals from the same subject are quantified.
 7. The method of claim 1, wherein the diagnostic sample is depleted of albumin and IgG before subjecting the diagnostic sample to two dimensional gel electrophoresis.
 8. The method of claim 7, wherein the tissue is plasma.
 9. The method of claim 1, comprising analyzing spots from the stained gel using liquid chromatography and mass spectrometry.
 10. The method of claim 8, wherein the mass spectrometry is SELDI mass spectrometry. 